EP1471140A1 - Liquide permettant le stockage a l'etat congele de cellules souches embryonnaires de primates et procede de stockage par congelation - Google Patents

Liquide permettant le stockage a l'etat congele de cellules souches embryonnaires de primates et procede de stockage par congelation Download PDF

Info

Publication number
EP1471140A1
EP1471140A1 EP03734902A EP03734902A EP1471140A1 EP 1471140 A1 EP1471140 A1 EP 1471140A1 EP 03734902 A EP03734902 A EP 03734902A EP 03734902 A EP03734902 A EP 03734902A EP 1471140 A1 EP1471140 A1 EP 1471140A1
Authority
EP
European Patent Office
Prior art keywords
cryopreservation
cryopreservation medium
cells
serum
cryoprotectant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03734902A
Other languages
German (de)
English (en)
Other versions
EP1471140A4 (fr
Inventor
Norio Nakatsuji
Hirofumi Pearl Haitsu Inari 3001 SUEMORI
Isao Asahi Techno Glass Corporation ASAKA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AGC Techno Glass Co Ltd
Original Assignee
Asahi Techno Glass Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Asahi Techno Glass Corp filed Critical Asahi Techno Glass Corp
Publication of EP1471140A1 publication Critical patent/EP1471140A1/fr
Publication of EP1471140A4 publication Critical patent/EP1471140A4/fr
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]

Definitions

  • the present invention relates to a cryopreservation medium and a cryopreservation method for embryonic stem (ES) cells from primates including humans, which have the potential to differentiate into any tissues in the body and promising applications in the fields of cell culture, tissue transplantation, research for drug discovery and gene therapy.
  • ES embryonic stem
  • Embryonic stem cells are cell lines derived from the inner cell mass of the blastocyst. When undifferentiated, they are pluripotent and can contribute to formation of any tissues including the germ line. Experiments using animals such as mice showed that blastocysts or morulae having ES cells injected therein or morulae with clumps of ES cells attached (Wood, S.A. et al., Proc. Natl. Acad. Sci. USA 90:4582-4585 (1993)) develop into progeny having two different genomes (which is called chimeric progeny). Moreover, ES cells are used in numerous medical studies to develop animal models of human diseases (Smithies, O. et al., Proc.
  • Suemori et al. succeeded in establishment of ES cell lines from intracytoplasmic sperm injected ova of cynomolgus monkeys, which are used widely in medical studies for their usefulness in preclinical testing, and demonstrated that they retain their pluripotency for a long term (2001 Dev. Dyn. Oct; 222(2):273-9; and JP-A-2002-159289).
  • the potency of the cynomolgus monkey ES cells to differentiate to dopaminergic neurons was also confirmed (2002 Proc. Natl. Acad. Sci. USA 99:1580-1585).
  • the ES cells of primates including humans established by the above method have a problem of poor cryopreservation efficiency under the conventional cryopreservation conditions mainly used for mouse ES cells. Therefore, when frozen according to conventional protocols, they take at least 2 to 4 weeks to proliferate in culture enough to be subcultured.
  • cryopreservation composition for nucleated cells including hematopoietic stem cells (JP-A-2000-201672) has already been reported.
  • a method applicable to primate ES cells has not been reported yet.
  • the object of the present invention is to provide a cryopreservation medium and a cryopreservation method which make it possible to cryopreserve ES cells from primates including humans, if not known yet, simply with high viability.
  • the present inventors have carried out studies to solve the above-mentioned problems and have completed the present invention on the basis of the finding that the use of a cryopreservation medium containing a cryoprotectant at a higher concentration than ever, specifically at a concentration of from 12% (W/V) to 50% (W/V), allows cryopreservation of primate ES cells with easy control at low cost.
  • the present invention provides the following invention.
  • the cryopreservation medium useful for cryopreservation of primate embryonic stem cells of the present invention preferably contains at least one member selected from dimethyl sulfoxide (hereinafter referred to simply as DMSO), glycerin, ethylene glycol, propylene glycol and polyvinyl pyrrolidone as a cryoprotectant. These may be contained singly or in combination.
  • DMSO dimethyl sulfoxide
  • glycerin ethylene glycol
  • propylene glycol propylene glycol
  • polyvinyl pyrrolidone polyvinyl pyrrolidone
  • the concentration of the cryoprotectant which ensures the effects of the present invention is from 12% (W/V) to 50% (W/V), suitably from 15% (W/V) to 30% (W/V).
  • the values are selected by the balance between cryoprotection effects and toxicity, and their approximations are also covered by the present invention as far as they meet the object of the present invention.
  • cryoprotection effects are weak at too low a concentration, while because of the reports that embryonic stem cells and embryonic carcinoma cells in culture begin to differentiate in 4 days at a DMSO concentration of above 1% (W/V) (Nicholas et al., Development (1994) 120: 1473-1482, Steven, Development (1994) 120: 3301-3312) and the possibility of carryover contamination of cell cultures, it is not preferred to use the cryoprotectant at an excessively high concentration.
  • the concentration in the cryopreservation medium is suitably from 15% (W/V) to 30% (W/V), particularly suitably from 15% (W/V) to 25% (W/V) (Fig. 18 and Fig. 19).
  • the concentration in the cryopreservation medium is particularly suitably from 12% (W/V) to 30% (W/V) (Figs. 20 to 22).
  • JP-A-2000-201672 discloses a cryopreservation composition containing from 3% (W/V) to 20% (W/V) of DMSO in combination with dextran and albumin.
  • it merely refers to the conventionally used maximum and minimum concentrations ("Soshiki Baiyo no Gijutsu” edited by the Japanese Tissue Culture Association (1982) p.42-44, "Shin-Seikagaku Jikken Koza vol.
  • DMSO is considered to be harmful at concentrations of 10% or above ("Bunshi-Saibo-Seibutsugaku Jiten” edited by Muramatsu Masami et al. (1997) p.391) so far, and experiments using chicken lung cells did not give high viabilities at DMSO or glycerin concentrations as high as from 15 to 20% (Dougherty, R, M. Nature 193:550(1962)). Therefore, successful cryopreservation of primate embryonic stem cells at high cryoprotectant concentrations as in the present invention is a totally new finding.
  • the cryopreservation medium of the present invention suitably contains serum and/or a serum replacement at a concentration of from 10% (W/V) to 85% (W/V).
  • FBS is suitably used as the serum.
  • the serum replacement suitably contains at least one component selected from the group consisting of albumin or albumin substitutes, transferrin or transferrin substitutes, and insulin or insulin substitutes.
  • a serum replacement is used to maintain embryonic stem cells in an undifferentiated stage. It seems that serum and/or a serum replacement is essential at least at the cryopreservation. Excluding the cryoprotectant, serum or a serum replacement may constitute the rest of the cryopreservation medium of the present invention.
  • cryopreservation medium is preferred to contain serum and/or a serum replacement at a low concentration (Figs. 25 and 26).
  • a final concentration of at least 10% (W/V) seems necessary to ensure cell stabilization.
  • the particularly suitable concentration of serum and/or a serum replacement is from 10% (W/V) to 50% (W/V).
  • a serum replacement containing at least albumin or albumin substitutes is suitable.
  • a serum replacement containing at least two components, albumin or albumin substitutes and transferrin or transferrin substitutes is particularly suitable.
  • the optimum serum replacement contains at least three components, albumin or albumin substitutes, transferrin or transferrin substitutes, and insulin or insulin substitutes.
  • the serum replacement further contains at least one component selected from the group consisting of amino acids, vitamins, antioxidants, collagen precursors and trace elements. Serum may be supplemented with these components, if necessary.
  • the albumin is suitably bovine serum albumin (BSA), fetal bovine albumin (fetuin), human serum albumin (HAS), a recombinant human albumin (rHSA), ovalbumin or the like.
  • the albumin substitutes are suitably bovine pituitary extract, bovine embryo extract, chicken extract, vegetable albumin or the like.
  • the transferrin is suitably iron-bound transferrin or iron-free transferrin. In the latter case, coexistence of iron ions is suitable.
  • the transferrin substitutes are suitably a soluble iron compound or an iron ion.
  • iron chelate compounds such as the ferric citrate chelate or the ferrous sulfate chelate is preferred.
  • an insulin bound to a metal ion such as the zinc ion or metal-free insulin is suitable. In the latter case, coexistence of a metal ion is suitable.
  • a soluble metal compound or a metal ion is suitable.
  • zinc is suitable. Zinc chloride, zinc bromide or zinc sulfide heptahydrate may specifically be mentioned.
  • amino acids glycine, L-alanine, L-asparagine, L-cysteine, L-asparaginic acid, L-glutaminic acid, L-phenylalanine, L-histidine, L-isoleusine, L-lysine, L-leucine, L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline and L-valine may be mentioned.
  • vitamins in the vitamin B group such as biotin, pantothenic acid, choline, folacin (folic acid), myoinositol, niacin, pyridoxine, riboflavin, thiamine and cobalamin are suitable.
  • antioxidants glutathione and ascorbic acid may be mentioned.
  • collagen precursors a poly(L-proline) or its derivatives, or a poly(L-hydroxyproline) or its derivatives may be mentioned.
  • components such as amino acids, vitamins, antioxidants, collagen precursors and trace elements may be used in combinations, of two or more of the same type or different types.
  • the cryopreservation medium of the present invention may further contain a basal medium for animal tissue culture at a concentration of from 10% (W/V) to 75% (W/V).
  • a basal medium for animal tissue culture containing at least from 0.3% (W/V) to 5% (W/V) of glucose is suitably used at a concentration of from 10% (W/V) to 75% (W/V) because of recovery of thawed cells and suppression of apoptosis.
  • the selected glucose content reflects the fact that though animal tissue culture media are usually used at a minimum glucose concentration of about 0.1%, media with slightly higher glucose content are usually used for embryonic stem cells.
  • a basal medium suitable for culture of the cells is incorporated in the cryopreservation medium.
  • a basal medium is added so that the concentration of the cryoprotectant and serum and/or the serum replacement is the essential minimum (about 25% (W/V) in total), while the basal medium constitutes the other 75% (W/V).
  • W/V the concentration of the cryoprotectant and serum and/or the serum replacement
  • the basal medium constitutes the other 75% (W/V).
  • the minimum amount of it necessary to obtain its effect is 10% (W/V).
  • the above-mentioned cryoprotectant is used to cryopreserve harvested primate embryonic stem cells, namely, by a method comprising a step of suspending primate embryonic stem cells in a cryopreservation medium containing the cryoprotectant at a concentration of from 12% (W/V) to 50% (W/V), a refrigeration step of freezing the suspension of the primate embryonic stem cells in the cryopreservation medium by cooling it to -80°C or below, and a preservation step of storing the frozen suspension of primate embryonic stem cells in the cryopreservation medium.
  • the cooling rate is preferably from 0.5°C to 10°C per 1 minute, because rapid cooling leads to difference in ice formation between the intracellular water and the extracellular water which is destructive of the cellular microstructure.
  • the preservation step can be performed at -80°C or below preferably in liquid nitrogen and/or liquid nitrogen vapor for more stable storage.
  • the details of the mechanism underlying the present invention are not clear.
  • primate embryonic stem cells are supposed to be more susceptible to cryodamage than mouse embryonic stem cells, it is speculated that the use of a cryoprotectant at higher concentrations than usual suppresses cryodamage and remarkably improves cell viability.
  • the present invention provides a cryopreservation medium and a cryopreservation method which allow simple cryopreservation of primate embryonic stem cells with high viability.
  • Cynomolgus monkey ES cells were subcultured for 33 passages and dissociated into cell suspension from three 90-mm Petri dishes, and the cell suspension was centrifuged at 1,000 rpm for 5 minutes. The supernatant was removed to obtain a cell precipitate.
  • the cell suspension was transferred to a serum tube, refrigerated to -80°C at a rate of about 1°C/min in Mr. Frosty (Nalgen) and then stored in a liquid nitrogen container for about 6 days.
  • a cell suspension in 1 ml of a cryopreservation medium consisting of 0% (V/V) of DMSO, 50% (V/V) of serum replacement (A) and 50% (V/V) of medium (A) was prepared, transferred to a serum tube, refrigerated to -80°C at a rate of about 1°C/min in Mr. Frosty (Nalgen) and then stored in a liquid nitrogen container for about 6 days.
  • the cells were thawed, plated in 60-mm Petri dishes in duplicate and cultured in the medium described in Suemori et al. (2001 Dev. Dyn. Oct; 222(2):273-9).
  • ES cell proliferation was investigated by following the time course of the activity of alkaline phosphatase, which is a marker of ES cells. After 3, 5 and 7 days, the medium was removed from the dishes by aspiration, and the cells were washed with 1 ml of 30 mM Hepes buffer containing 0.1 mg/l of CaCl 2 and 0.1 mg/l of MgSO 4 (7H 2 O) and treated with 1 ml of 0.2 mM 4-methylumbelliferyl phosphate solution in the same buffer in an incubator at 37°C for 1 hour. 0.8 ml portions of the reaction mixture were transferred to a 24-well microplate and analyzed by fluorometry at Ex355/Em460. Fluorescence per 1 ml was calculated and plotted. As a control, the alkaline phosphatase activity of the same feeder cells as used during subcultures of ES cells was measured.
  • the cells in the illustrative experiment showed significantly higher alkaline phosphatase activity than the feeder cells (Fig. 1) and could be subcultured after 1 week. After three-fold dilution followed by 3 days of subculture, successful proliferation of ES cells was observed without formation of differentiated colonies (Figs. 2 and 3). After completion of the culture, the cells in each Petri dish were fixed with paraformaldehyde and ethanol and stained with Vector Red alkaline phosphatase substrate (Vector Labs). The numerous successfully stained colonies (Fig. 4) demonstrate that the ES cells cryopreserved according to the present invention remained normal.
  • Cynomolgus monkey ES cells subcultured for 34 passages were harvested from six 90-mm Petri dishes and treated by a method similar to that employed in Example 1 to give a cell precipitate.
  • the cell suspension was transferred to two serum tubes, refrigerated to -80°C at a rate of about 1°C/min in Mr. Frosty (Nalgen) and stored in a liquid nitrogen container for 1 day.
  • a cell suspension in 2 ml of a cryopreservation medium consisting of 10% (V/V) of DMSO, 45% (V/V) of serum replacement (A) and 45% (V/V) of medium (A) was prepared, transferred to two serum tubes, refrigerated to -80°C at a rate of about 1°C/min in Mr. Frosty (Nalgen) and then stored in a liquid nitrogen container for 1 day.
  • a cryopreservation medium consisting of 10% (V/V) of DMSO, 45% (V/V) of serum replacement (A) and 45% (V/V) of medium (A) was prepared, transferred to two serum tubes, refrigerated to -80°C at a rate of about 1°C/min in Mr. Frosty (Nalgen) and then stored in a liquid nitrogen container for 1 day.
  • the cells in each serum tube were thawed, plated in 60-mm Petri dishes in duplicate and cultured in the medium described in Suemori et al. (2001 Dev. Dyn. Oct;222(2):273-9).
  • ES cell proliferation was assessed by alkaline phosphatase activity as described in Example 1.
  • the cells in the illustrative experiment showed significantly higher alkaline phosphatase activity than the feeder cells (Fig. 5) and the cultures reached the stage at which they were able to be subcultured after 5 days (Figs. 6 and 7). After 7 days of prolonged culture, successful proliferation of ES cells was observed without formation of differentiated colonies (Figs. 8 and 9).
  • Cynomolgus monkey ES cells subcultured for 35 passages were harvested from three 90-mm Petri dishes and treated by a method similar to that employed in Example 1 to give a cell precipitate.
  • the cell suspension was transferred to a serum tube, refrigerated to -80°C at a rate of about 1°C/min in Mr. Frosty (Nalgen) and stored in a liquid nitrogen container for 5 day.
  • the cells were thawed and continuously subcultured in duplicate in 35-mm Petri dishes in the medium described in Suemori et al. (2001 Dev. Dyn. Oct;222(2):273-9).
  • Differentiation potency was assessed after 10 passages.
  • the cells were dissociated from the Petri dishes, and 5 ⁇ 10 6 cells were suspended in 0.5 ml of a phosphate buffer and intraperitoneally injected into SCID mice.
  • the mice were fed for about 6 weeks, and teratomas were isolated from the abdomen. Observation of the teratomas after Bouin fixation, paraffin sectioning and sematoxylin ⁇ eosin staining revealed formation of nerves, muscles and cartilage and demonstrated retention of pluripotency (Fig. 17).
  • Cynomolgus monkey ES cells subcultured for 35 passages were harvested from forty-eight 60-mm Petri dishes each carrying 4 ⁇ 10 6 cells and treated by a method similar to that employed in Example 1 to give a cell precipitate.
  • the cells were thawed, plated in 60-mm Petri dishes in duplicate and cultured in the medium described in Suemori et al. (2001 Dev. Dyn. Oct;222(2):273-9).
  • ES cell proliferation was assessed by alkaline phosphatase assay as described in Example 1.
  • the cells on each Petri dish was dissociated by tripsinization and counted.
  • the time-dependent increase in alkaline phosphatase activity indicates successful.
  • Fig. 16 indicates that DMSO concentrations between 15% (W/V) and 25% (W/V) were preferred.
  • Cynomolgus monkey ES cells subcultured for 32 passages were harvested from thirty-two 60-mm Petri dishes each carrying 2.8 ⁇ 10 6 cells and treated by a method similar to that employed in Example 1 to give a cell precipitate.
  • ES cell proliferation was assessed by alkaline phosphatase assay as described in Example 1.
  • Cynomolgus monkey ES cells subcultured for 41 passages were harvested from fifty-six 60-mm Petri dishes each carrying 2.5 ⁇ 10 6 cells and treated by a method similar to that employed in Example 1 to give a cell precipitate.
  • ES cell proliferation was assessed by alkaline phosphatase assay as described in Example 1.
  • the cells on each Petri dish was dissociated by tripsinization and counted.
  • alkaline phosphatase activity increased faster at any cell densities than in the comparative experiment (Fig. 23).
  • the cell count per Petri dish after 8 days of culture was greater than that in the comparative experiment, though tends to increase with the storage cell density, which indicates that increase in cryoprotectant concentration has greater effect than increase in freezing cell density (Fig. 24).
  • Cynomolgus monkey ES cells subcultured for 45 passages were harvested from forty-eight 60-mm Petri dishes each carrying 4.0 ⁇ 10 6 cells and treated by a method similar to that employed in Example 1 to give a cell precipitate.
  • ES cell proliferation was assessed by alkaline phosphatase assay as described in Example 1.
  • the cells on each Petri dish was dissociated by tripsinization and counted.
  • alkaline phosphatase activity increased (Fig. 25)
  • the cell counting per Petri dish after 7 days of culture gave similar results (Fig. 26).
  • Successful cryopreservation was confirmed at any supplement concentrations, though cryopreservation efficiency tended to be better at low supplement concentrations, irrespective of the kind of supplement.
  • Cynomolgus monkey ES cells subcultured for 32 passages were harvested from thirty-two 60-mm Petri dishes each carrying 2.8 ⁇ 10 6 cells and treated by a method similar to that employed in Example 1 to give a cell precipitate.
  • the present invention enables simple cryopreservation of primate ES cells with high viability. Cryopreservation using a serum replacement containing albumin, insulin and transferrin can prevent differentiation induced by unknown factors in serum. Besides, because all the procedures from culture through storage can be carried out without sera, use of ES cells obtained by the present invention in regenerative medicine and gene therapy can obviate risks of contamination by unknown pathogens in sera.
EP03734902A 2002-01-31 2003-01-31 Liquide permettant le stockage a l'etat congele de cellules souches embryonnaires de primates et procede de stockage par congelation Withdrawn EP1471140A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2002024382 2002-01-31
JP2002024382 2002-01-31
PCT/JP2003/000999 WO2003064634A1 (fr) 2002-01-31 2003-01-31 Liquide permettant le stockage a l'etat congele de cellules souches embryonnaires de primates et procede de stockage par congelation

Publications (2)

Publication Number Publication Date
EP1471140A1 true EP1471140A1 (fr) 2004-10-27
EP1471140A4 EP1471140A4 (fr) 2005-02-16

Family

ID=27654483

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03734902A Withdrawn EP1471140A4 (fr) 2002-01-31 2003-01-31 Liquide permettant le stockage a l'etat congele de cellules souches embryonnaires de primates et procede de stockage par congelation

Country Status (7)

Country Link
US (1) US20050026133A1 (fr)
EP (1) EP1471140A4 (fr)
JP (1) JP4223961B2 (fr)
AU (1) AU2003238620B2 (fr)
CA (1) CA2474968A1 (fr)
NZ (1) NZ534428A (fr)
WO (1) WO2003064634A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007015252A3 (fr) * 2005-08-03 2007-08-02 Imt Interface Multigrad Tech Ltd Cellules somatiques utilisees dans la therapie cellulaire
US7892726B2 (en) 2004-06-07 2011-02-22 Core Dynamics Limited Method for sterilizing lyophilized eukaryotic anuclear cells with gamma irradiation
US7935478B2 (en) 2004-02-02 2011-05-03 Core Dynamics Limited Biological material and methods and solutions for preservation thereof
US8037696B2 (en) 2004-08-12 2011-10-18 Core Dynamics Limited Method and apparatus for freezing or thawing of a biological material
WO2010096821A3 (fr) * 2009-02-23 2011-11-17 Cell & Tissue Systems, Inc. Procédé de cryoconservation sans glace d'un tissu
US8196416B2 (en) 2004-02-02 2012-06-12 Core Dynamics Limited Device for directional cooling of biological matter
US8480757B2 (en) 2005-08-26 2013-07-09 Zimmer, Inc. Implants and methods for repair, replacement and treatment of disease
US8497121B2 (en) 2006-12-20 2013-07-30 Zimmer Orthobiologics, Inc. Method of obtaining viable small tissue particles and use for tissue repair
US8518433B2 (en) 2003-12-11 2013-08-27 Zimmer, Inc. Method of treating an osteochondral defect
US9138318B2 (en) 2007-04-12 2015-09-22 Zimmer, Inc. Apparatus for forming an implant
WO2015151047A1 (fr) * 2014-04-03 2015-10-08 Biopredic International Technique de cryoconservation sans centrifugation et produits ainsi obtenus
US10167447B2 (en) 2012-12-21 2019-01-01 Zimmer, Inc. Supports and methods for promoting integration of cartilage tissue explants

Families Citing this family (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0896825B1 (fr) * 1997-08-14 2002-07-17 Sulzer Innotec Ag Composition et dispositif pour la réparation de cartilage in vivo comprenant des nanocapsules avec des facteurs ostéoinductifs et/ou chondroinductifs
US7622562B2 (en) 2002-06-26 2009-11-24 Zimmer Orthobiologics, Inc. Rapid isolation of osteoinductive protein mixtures from mammalian bone tissue
WO2005045007A1 (fr) * 2003-11-06 2005-05-19 Kyoto University Procede et systeme pour stocker des cellules souches a l'etat congele
US20090227032A1 (en) * 2005-12-13 2009-09-10 Kyoto University Nuclear reprogramming factor and induced pluripotent stem cells
EP2206724A1 (fr) * 2005-12-13 2010-07-14 Kyoto University Facteur de reprogrammation nucléaire
US8129187B2 (en) * 2005-12-13 2012-03-06 Kyoto University Somatic cell reprogramming by retroviral vectors encoding Oct3/4. Klf4, c-Myc and Sox2
US8278104B2 (en) * 2005-12-13 2012-10-02 Kyoto University Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2
US20080154233A1 (en) * 2006-12-20 2008-06-26 Zimmer Orthobiologics, Inc. Apparatus for delivering a biocompatible material to a surgical site and method of using same
US9213999B2 (en) * 2007-06-15 2015-12-15 Kyoto University Providing iPSCs to a customer
JP2008307007A (ja) * 2007-06-15 2008-12-25 Bayer Schering Pharma Ag 出生後のヒト組織由来未分化幹細胞から誘導したヒト多能性幹細胞
SG10201400329YA (en) 2008-05-02 2014-05-29 Univ Kyoto Method of nuclear reprogramming
CN102741396B (zh) * 2009-04-27 2017-10-20 威盛细胞有限公司 支持多能细胞生长的小分子和其方法
US20150024406A1 (en) * 2013-07-17 2015-01-22 Robin N. Coger-Simmons Increased aquaporin expression on cellular membrane to improve cryopreservation efficiency
WO2016007506A1 (fr) * 2014-07-07 2016-01-14 Targazyme, Inc. Production et cryopréservation de cellules fucosylées à usage thérapeutique
JP2016073207A (ja) * 2014-10-02 2016-05-12 株式会社リプロセル 細胞凍結保存方法、細胞解凍方法、及び細胞
CN106999518A (zh) * 2014-11-14 2017-08-01 日本赤十字社 脐带血以及末梢血的冻结保存方法以及冻结保存用溶液
FR3040396A1 (fr) * 2015-06-30 2017-03-03 Chu Nantes Procede de cryoconservation de lymphocytes infiltrant la tumeur
FR3038325A1 (fr) * 2015-06-30 2017-01-06 Lab Francais Du Fractionnement Procede de cryoconservation de cellules a visee therapeutique
FR3038324B1 (fr) * 2015-06-30 2020-10-30 Lab Francais Du Fractionnement Procede de cryoconservation de cellules a visee therapeutique
JP6727334B2 (ja) * 2016-02-01 2020-07-22 グリーン・クロス・ラブ・セル・コーポレイション 細胞の凍結保存のための培地組成物およびその使用
WO2017196175A1 (fr) 2016-05-12 2017-11-16 Erasmus University Medical Center Rotterdam Procédé de culture de cellules myogènes, cultures ainsi obtenues, procédés de criblage et milieu de culture cellulaire
JP6353615B1 (ja) 2016-12-14 2018-07-04 株式会社大塚製薬工場 哺乳動物細胞凍結保存液
CN107047541A (zh) * 2017-05-31 2017-08-18 东莞市保莱生物科技有限公司 一种免疫细胞冻存液及免疫细胞冻存方法
DK3644728T3 (da) * 2017-06-28 2022-10-31 Sci Group As Frysning af biologisk materiale
NL2019517B1 (en) 2017-09-08 2019-03-19 Univ Erasmus Med Ct Rotterdam New therapy for Pompe disease
CN108812645A (zh) * 2018-07-20 2018-11-16 吉林济惠生物科技有限公司 一种人脐带间充质干细胞的细胞冻存液
WO2020067434A1 (fr) * 2018-09-27 2020-04-02 テルモ株式会社 Méthode de cryoconservation de cellules
CN110199987A (zh) * 2019-07-22 2019-09-06 深圳市未来细胞生命科技有限公司 一种间充质干细胞保存液及其保存方法和应用
CN111226910B (zh) * 2020-03-09 2022-03-25 佛山辅康生物科技有限公司 卵子或卵裂期胚胎的玻璃化冷冻液及冷冻方法
CN115251038A (zh) * 2021-04-30 2022-11-01 中国科学院化学研究所 包含丝素蛋白的冷冻保存液及其应用和控冰冻存方法
CN113881622B (zh) * 2021-09-30 2023-12-15 齐国光 一种干细胞的保存运输方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4559298A (en) * 1982-11-23 1985-12-17 American National Red Cross Cryopreservation of biological materials in a non-frozen or vitreous state
WO1991003935A1 (fr) * 1989-09-22 1991-04-04 Tsi-Mason Research Institute Procede et compositions de cryoconservation en une etape d'embryons
WO2000019818A1 (fr) * 1998-10-07 2000-04-13 Cedars-Sinai Medical Center Procede de conditionnement et de cryopreservation de cellules
EP1347040A1 (fr) * 2000-12-04 2003-09-24 Humantec Ltd. Liquide de conservation de cellules et procede de conservation de cellules dans lequel ledit liquide est utilise

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3316887A1 (de) * 1983-05-07 1984-11-08 Agfa-Gevaert Ag, 5090 Leverkusen Photographisches aufzeichnungsmaterial und neue azofarbstoffe
JPS6360931A (ja) * 1986-08-29 1988-03-17 Noboru Sato 血液または血液製剤保存液およびこれを用いた血液または血液製剤の保存方法
IT1207525B (it) * 1987-06-23 1989-05-25 Ist Naz Ric Sul Cancro Metodo per la preservazione difogli trapiantabili di epitelio coltivato in vitro vitale.
US6007978A (en) * 1988-05-18 1999-12-28 Cobe Laboratories, Inc. Method of freezing cells and cell-like materials
BR9003310A (pt) * 1990-07-10 1992-01-28 Brasil Pesquisa Agropec Processo e composicao para a criopreservacao de embrioes de mamiferos
US6071889A (en) * 1991-07-08 2000-06-06 Neurospheres Holdings Ltd. In vivo genetic modification of growth factor-responsive neural precursor cells
US5843780A (en) * 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
US6667176B1 (en) * 2000-01-11 2003-12-23 Geron Corporation cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells
JP2000201672A (ja) * 1999-01-11 2000-07-25 Asahi Medical Co Ltd 有核細胞の凍結保存用組成物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4559298A (en) * 1982-11-23 1985-12-17 American National Red Cross Cryopreservation of biological materials in a non-frozen or vitreous state
WO1991003935A1 (fr) * 1989-09-22 1991-04-04 Tsi-Mason Research Institute Procede et compositions de cryoconservation en une etape d'embryons
WO2000019818A1 (fr) * 1998-10-07 2000-04-13 Cedars-Sinai Medical Center Procede de conditionnement et de cryopreservation de cellules
EP1347040A1 (fr) * 2000-12-04 2003-09-24 Humantec Ltd. Liquide de conservation de cellules et procede de conservation de cellules dans lequel ledit liquide est utilise

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1 April 2000 (2000-04-01), DHALI A ET AL: "Vitrification of buffalo (Bubalus bubalis) oocytes" XP002310885 Database accession no. PREV200000264997 & THERIOGENOLOGY, vol. 53, no. 6, 1 April 2000 (2000-04-01), pages 1295-1303, ISSN: 0093-691X *
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1987, SANDLER S ET AL: "CRYOPRESERVATION OF MOUSE PANCREATIC ISLETS EFFECTS OF DIFFERENT GLUCOSE CONCENTRATIONS IN THE POST-THAW CULTURE MEDIUM ON ISLET RECOVERY" XP002310884 Database accession no. PREV198784086495 & CRYOBIOLOGY, vol. 24, no. 4, 1987, pages 285-291, ISSN: 0011-2240 *
DATABASE WPI Section Ch, Week 199209 Derwent Publications Ltd., London, GB; Class D22, AN 1992-065217 XP002310886 & BR 9 003 310 A (EMBRAPA EMPRESA BRA) 28 January 1992 (1992-01-28) *
See also references of WO03064634A1 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8518433B2 (en) 2003-12-11 2013-08-27 Zimmer, Inc. Method of treating an osteochondral defect
US8834914B2 (en) 2003-12-11 2014-09-16 Zimmer, Inc. Treatment methods using a particulate cadaveric allogenic juvenile cartilage particles
US8784863B2 (en) 2003-12-11 2014-07-22 Zimmer, Inc. Particulate cadaveric allogenic cartilage system
US8765165B2 (en) 2003-12-11 2014-07-01 Zimmer, Inc. Particulate cartilage system
US8652507B2 (en) 2003-12-11 2014-02-18 Zimmer, Inc. Juvenile cartilage composition
US8524268B2 (en) 2003-12-11 2013-09-03 Zimmer, Inc. Cadaveric allogenic human juvenile cartilage implant
US8512941B2 (en) 2004-02-02 2013-08-20 Core Dynamics Limited Biological material and methods and solutions for preservation thereof
US8196416B2 (en) 2004-02-02 2012-06-12 Core Dynamics Limited Device for directional cooling of biological matter
US7935478B2 (en) 2004-02-02 2011-05-03 Core Dynamics Limited Biological material and methods and solutions for preservation thereof
US7892726B2 (en) 2004-06-07 2011-02-22 Core Dynamics Limited Method for sterilizing lyophilized eukaryotic anuclear cells with gamma irradiation
US8037696B2 (en) 2004-08-12 2011-10-18 Core Dynamics Limited Method and apparatus for freezing or thawing of a biological material
WO2007015252A3 (fr) * 2005-08-03 2007-08-02 Imt Interface Multigrad Tech Ltd Cellules somatiques utilisees dans la therapie cellulaire
US8198085B2 (en) 2005-08-03 2012-06-12 Core Dynamics Limited Somatic cells for use in cell therapy
US8480757B2 (en) 2005-08-26 2013-07-09 Zimmer, Inc. Implants and methods for repair, replacement and treatment of disease
US8497121B2 (en) 2006-12-20 2013-07-30 Zimmer Orthobiologics, Inc. Method of obtaining viable small tissue particles and use for tissue repair
US9138318B2 (en) 2007-04-12 2015-09-22 Zimmer, Inc. Apparatus for forming an implant
WO2010096821A3 (fr) * 2009-02-23 2011-11-17 Cell & Tissue Systems, Inc. Procédé de cryoconservation sans glace d'un tissu
US8440390B2 (en) 2009-02-23 2013-05-14 Lifeline Scientific, Inc. Method for ice-free cryopreservation of tissue
US10167447B2 (en) 2012-12-21 2019-01-01 Zimmer, Inc. Supports and methods for promoting integration of cartilage tissue explants
WO2015151047A1 (fr) * 2014-04-03 2015-10-08 Biopredic International Technique de cryoconservation sans centrifugation et produits ainsi obtenus
US10292382B2 (en) 2014-04-03 2019-05-21 Biopredic International Sarl No-spin cryopreservation technique and resulting products

Also Published As

Publication number Publication date
EP1471140A4 (fr) 2005-02-16
US20050026133A1 (en) 2005-02-03
JPWO2003064634A1 (ja) 2005-05-26
NZ534428A (en) 2006-12-22
AU2003238620B2 (en) 2008-11-06
CA2474968A1 (fr) 2003-08-07
WO2003064634A1 (fr) 2003-08-07
JP4223961B2 (ja) 2009-02-12

Similar Documents

Publication Publication Date Title
EP1471140A1 (fr) Liquide permettant le stockage a l'etat congele de cellules souches embryonnaires de primates et procede de stockage par congelation
US10472606B2 (en) Cell preservation method for pluripotent stem cells
US20180153155A1 (en) Method for cryopreservation of cardiocytes derived from pluripotent stem cells or mesenchymal stem cells derived from adipose tissue or bone marrow
US11540507B2 (en) Solution for cryopreservation of animal cells or animal tissues, cryopreserved product, and cryopreservation method
Seo et al. Cryopreservation of amniotic fluid-derived stem cells using natural cryoprotectants and low concentrations of dimethylsulfoxide
JP5998265B2 (ja) トレハロース及びデキストラン含有哺乳動物細胞移植用溶液
US20050106554A1 (en) Cryopreservation of pluripotent stem cells
US20130267008A1 (en) Freezing medium composition for cryopreserving amniotic fluid-derived stem cells and a method for cryopreserving the same
AU2017377309B9 (en) Mammalian cell cryopreservation liquid
KR102391629B1 (ko) 펙틴과 알라닌을 이용한 세포 동결 보존용 조성물 및 이를 이용한 세포 동결 보존 방법
US20220394971A1 (en) Methods and compositions for cryopreservation of cell therapies
US7501231B2 (en) Methods and compositions for cryopreservation of dissociated primary animal cells
JP6487552B2 (ja) 植物由来組換えヒト血清アルブミン及び植物性ペプチドを有効成分として含有する細胞保存用組成物
JP5590821B2 (ja) 多能性幹細胞用の凍結保存液と多能性幹細胞の凍結保存方法
CN112544613A (zh) 一种多能干细胞冻存液、其应用及冻存方法
JP2013236558A (ja) 細胞増殖抑制剤
Ock et al. Isolation and viability of presumptive spermatids collected from bull testes by Percoll density gradient
Matsumura et al. Hypothermicpreservation of Mouse Induced Pluripotent Stem Cells by Polyampholytes
Abrafikova et al. Hypothermic Storage and Cryopreservation in Technology of Obtaining Human Skin Fibroblasts

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20040805

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO

RIC1 Information provided on ipc code assigned before grant

Ipc: 7A 01N 1/02 B

Ipc: 7C 12N 5/06 B

Ipc: 7C 12N 5/02 A

A4 Supplementary search report drawn up and despatched

Effective date: 20050104

17Q First examination report despatched

Effective date: 20070403

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20080927